Butyl alcohol and acetone fermentation process



Patented Ma 19, 1925.

UNITED STATES PATEN Ti OFFICE.

FOREST J. FUNK, OF WILMINGTON, DELAWARE, .ASSIGNOR TO E. I. DU PONLI DE.NEMOURS & COMPANY, or WARE.

No Drawing.

7 tion of carbohydrates, and especially of the sugars occurring inmolasses, and pertains more particularly to fermentations which arebrought about by a micro-organism which will be hereinafter designatedbacillus aoeto-butylicum.

Upon an investigation of the fermentation of molasses by bacillusaceto-butylicum it was found that, although satisfactory yields ofbutyl'alcohol and acetone could be obtained when working on a smallscale,- large scale fermentations could i not be carried out withuniformly successful results, the yield of butyl alcohol and acetonesometimes being satisfactory, but more often quite unsatisfactory. Itwas finally found that the trouble was in all probability due to apersistent contamination in the form of an organism or organismsremarkably resistant to heat. The failure to detect sooner thiscontaminating organism, which apparently is present in all molasses, wasdue to two main peculiarities: First, the fact that its activities areaccompanied by no visible phenomena such as evolution of gas, and,second, that it is so small as to be.easily missed in a microscopicexamination. The discovery of this contaminating organism servedat'first only to explain past failures, and for some time there was nosure way of overcoming the difiiculties which it involved. Thiscondition of affairs was due to the fact that the organism is remarkablyresistant to heat, and thatin order. to kill it completely it wasnecessary to employ such a high temperature that the sugar in I themolasses was sufliciently caramelized to make it unsatlsfactory for use.

In my application Serial No. 414,087 filed, October 1, 1920, I havedescribed .and claimed anew seeding procedure by means of which I havebeen able to secure consistently good yields of butyl alcoholand aceto en. large scale operations from WILMINGTON, DELAWARE, A CORPORATION OFDELA- BUTYL ALCOHOL AND ACETONE FERMENTATION EROCES S.

Application filed February 21, 1921. Serial No. 446,764.

mashes containing black strap molasses as the source of sugar, ,Wltll orwithout corn gluten as an addltlonal nitrogen source.

The essential points of the procedure.

covered in my above-mentioned application cons1st, first, in developingthe seed culture in such a manner as will result in the great majorityof the butyl organisms being in the spore format the time the seed isused to inoculate the mash; and, second, in inocu lating the final mashat such a temperat-urethe temperature of pasteurization that theremaining vegetative cells will be destroyed and the spores themselvesso affected as to shorten their period of germination.-

I I have now discovered that the procedure described in myabove-mentioned application may be modified mainly Wit respect to,-

the second point set forth above, he modification being particularlyadvantageous in connection with large scale operations. Thismodification. broadly stated, consists essentially in inoculating thefinal mash at a temperature below 49 C., and preferably at thefermentation temperature of about 36.69 0., instead of inoculating thisfinal mash at the pasteurization temperature (about 63 0.). I have alsofound that the preliminary treatment of the seeding solution may becarried out in a manner some what different from that specificallydescribed in my prior application, but the differences here referred toare ofa minor character, the converslon of the mlcro-organisms in theseeding solution into-the spore form being still considered of primeimportance. 1

The main objects of the present invention, as Well'as of the inventioncovered in my prior-application, may be said to be the weakening of theheat-resistant contamination which I have found it impractical todestroy, and the activation of the spores of the micro-organism(particularly bacillus aceto-butylicum) so that they will germinate andproduce fermentation in the shortest possible time, after being broughtto the fermentation temperature.

The weakening of the heat-resistant contamination is accomplished to asufficient extent by subjecting the molasses mash, be-

fore the seed has been added, to the ordinary sterihzmg temperature, themash when sterilized being preferably a 50% solution of molasses. addedto the final mash while the latter is at a suitable fermentationtemperature (for example, 36.6 C.) a satisfactory fermentation withrespect to butyl alcohol and aceene blue.

tone production ensues.

The species of micro-organism which I prefer to use'has been namedbacillus 'aceto-. butylicum. The manner. of isolating this species ofbacteria will be hereinafter described, but in order to facilitate theiridentification the species is described at this point in accordance withthe descriptive chart oflthe Society of American Bacteriologists SourceGorn meal fromAmeric an maize. NameBac11lus aceto-butylicum.

I. Morphology.

1. Vegetable cells, motile: ,Media usednutrient agar slant containing 1%corn starch, or 5% corn media (1 part corn meal in 20 parts water),temp.

2 0., age 24 hours.

Formshort rods, ch 0 in formation. Size2-4 microns x 12 microns.Endsrounded. Stainevenly with Loefilers methylene lue or gentian violet.Gram stain positive.

2. Sporangia: Media used-nutrient agar slant containing 1% corn starch,temperature 32 C., after 2 days spores formed.

Form-oval. Spores central. Limits of size1.6 microns x 1.2 microns. Sizeof majority1.6 microns x 1.2 mi- CI'OIIS.

Spores stain poorly with Loefflers methyl- 1 I Cultural features.

If the activated seedbe then 2 II. Culturalfeatures.-Continued.

. Surface-smooth.

Elevationraised.

Edge-entire.

Internal structure-amorphous 11. Starch agar (1% corn starch in nutrientagar).

Growth-abundant.

Diastatic actionmarked.

17. Nitrogen source.

Proteins, peptone.

Ill. Physical and biochemical features.

reaction of media i For growth and fermentation, Sorensens Pvalues5.06.3.

8. Vitality on culture media. Several months at- 32 C. 9. Temperaturerelation. Optimum temperature 32 to 38 C. Spores resist 80 C. for 20minutes. 10. Resistant to drying. 13. Acids produced, butyric. 15.Alcohol, butyl. Ketone, acetone.

Isolation of the organisms.

After a careful investigation ofthe subject, the procedure adopted forisolating the bacillus aceto-butylicum was as follows:

Test tubes of corn meal solution are prepared, heated to 80 C. for abouttwenty minutes to kill the lessresistant bacteria, incubated at 32 C.Without removing the air, and then Watched closely for evidence of butylalcohol fermentation. The culture in the tube or tubes which shows anactive fermentation in the presence of air and yields a characteristicbutyl alcohol odor, are in part transferred to a solid agar cultureplate and the bacteria allowed to develop in colonies on its surface.Transfers are made from these colonies to fresh sterilized cornmealtubes and the type .noted which produced the characteristic may be alaboratory flask, at fifteen pounds steam pressure for about threehours. This mash is cooled to the fermentation temperature of 36.6 0.,and inoculated with an active culture of bacillus aceto-butylicum. Thiscorn meal mash is held at a temperatnre,of 36.6 0. for about two days,during l which time active fermentation sets in, the

gluten in the corn meal rises to the surface of the liquid; remainsthere during the height of the fermentation,v (meanwhile undergoingconsiderable alteration in its physical properties, becoming slimy andfull of entrained bubbles of gas evolved by the fermentation), afterwhich (it drops slowly to the bottom of the container; the period ofactive fermentation being over, the temperature is lowered to about 31"0., at which it is maintained for a further period of about five days,during which period, presumably owing to the exhaustion of the foodmaterials and the presence of the products of fermentation, thevegetative cells of the organism which have pro duced the fermentationrevert for the most part to the spore form, which is the condition inwhich they are used in my new procedure.

Shortly before tlns stage 1s reached a so lution 1s prepared consistingof twenty-six pnunds ofmolasses dissolved in about 3 gallens of water,to which is added about one and one-third pounds of commercial corngluten containing about 44% of protein. This mixture is sterilized byheating at from fifteen to twenty-five pounds steam pressure forone-half hour,- after which it is diluted to a final volume of fortygallons with watersterilized by heating, by ozone, or by ultra-violetlight; or it may be diluted with unsterilized water to the final volumeof forty gallons and again immediately sterilized by heating with orwithout pressure for a further period of one-half hour or more.

Up to this point the procedure is the same as that described in myapplication Ser. No. 414,087 mentioned above. Since the modificationsforming the subject matter of the present application are chieflyapplicable to cases where the final mash when fully diluted is at atemperature below 365 0., it will not be necessary in the remaindervofthis example to dwell upon thetreatment of a final mash which has beensterilized after dilution, or which has been prepared by diluting theconcentrated molasses solution with hot sterilized water.

Assuming that the dilution of the sterilized 50% molasses solution hasbeen made with sterile cold water, the resulting mash is'heated to afinal temperature of about 366 0. The seed culture. prepared as abovedescribed, is then handled in the fol lowingwvay: Not less than fifteenminutes before the complete final mash will be at the fermentationtemperature of 36.6 0., the seed culture is heated as rapidly aspossible to the pasteurization temperature, that is, between about '57and 77 0., for ex-' ample, to approximately 63 "0. This temperature ismaintained for fifteen minutes,

whereupon the heated seed, without previous cooling, is added as rapidlyas possible to the completed final mash. Two alternative methods ofheating the seed for pas teurization are possible, both of which-havebeen tried and found to give the sameresults. The first of these is toinject live steam directly into the seed mash itself un til the propertemperature is reached, and maintain this temperature by the injectionof further steam as may be required; the second method is to heat theseed mash indirectly, either by a steam coil in the mash or by anexternal water or steam .bath. Apparently the method of obtainingthenecessary temperature does not matter. The pasteurized seed is cooled tothe fermentation temperature instantly on mixing with the relativelylarge volume of the final mash, and fermentation follows the same courseas with my former regular procedure.

A second modification which I have devised diiiers from the one setforth in the above example in that the final sterile mash is brought toatemperature of about 46 0., the seed. added thereto without previouspasteurization, and the resulting inoculated mash maintained at thistemperature for several hours, the cooling of the mash being started atsuch a time as will permit of the temperature of the mash reaching36.60.

about five hours after the addition of the seed. The temperature of theinoculated minutes or more before the fermentation temperature isreached in the larger portion of the final mash, the spore seed, without previous pasteurization, is added to the fraction of the mash whichhas been heated to 63 C. and the resulting mixture maintained at about63 (lfor fifteen minutes. The fraction containing the seed, which hasbeen effectively pasteurized I by this heat treatment, is then added tothe main volume of the mat h. It is sometimes desirable to have thetemperature of the main portion of the mash a little below 366 (3., say333 (1., so that when the smaller portion, at a temperature of 63 C., isadded thereto the final mixture will be at a temperature of about 36.6C.

A fourth modification of the process, and one which may be.1'efer red toas a flash method. consists essentially in heating the seed quickly to atemperature substantially higher than the ordinary pasteurizationtemperature, as, for example, to a temperature of 8082.2 Q, and thenadding the seed, as soon as this high temperature has been reached,directly to the mash, the latter being at the fermentation temperature,366 0., or thereabouts. The following example illustrates how thisprocedure may be operated on a ommercial scale.

The molasses is sterilized in solution together with the co n gluten;then blown into the fermenter, which has previously been filled withsufficient sterile water to bring the total mash after the addition ofthe molasses, to the desired final volume and concentration. By means ofan inlet to the pipe conveying the molasses solution, the seed isadmitted into the stream. of hot -c ncentrated molasses at such a ratethat cation'of the apparatus required, the process described in myabovcmentioned application necessitating equipment for large scaleoperations. A considerable ,saving in heat energy is also effecteddispensing with the prelin'iinary step of heating the final mash to apasteurizing temperature of 63 C. or higher.

The underlying reasons for the adoption of the pasteurization proceduresof my present, as well as of my aforesaid prior, ap-

an elaborate cooling sources of sugar) contains a slightly motilorganism which is extremely resistant to heat, so much so that any steampressure xtreatment short of such a temperature as to caramelize thesugar and leave the molasses unfermentable, is insufiicient completelyto destroy this contamination. This organism produces non-volatile acidand is sufficiently active to inhibit the butyl fermentation if it gainsthe upper hand, It has very evidently been the cause of the failures andirregular results obtained prior to my invention.

In the second place I have found that by sterilizing the molasses in 50%solution an appreciably greater number of the organisms forming thiscontamination are destroyed than when the molasess is diluted to theconcentrationin which it is usually used in the final mash, that is, 6to 10%, before sterilization. It is important to carry out the variousmashing operations with as little delay as possible and to manipulatethe mash in such away during the entire period of preparation betweenthe initial sterilization and the final inoculation as to preclude thepossibility of any substantial recovery and growth of any survivingharmful organisms of the type described above.

In the third place, the effect of the temperature of pasteurization uponthe seed added is to destroy the remaining weakened vegetative cells andto act upon the spores in such a way that they germinate more readilythan is the case with the unheated spores, which usually germinaterather slowly. The germinating spores produce the new vegetative cellsin the medium which is to be fermented, and these cells are, therefore,better adapted to this medium than are those which have grown in amedium of some other constitution, as for example, corn meal mash. Inthis way I have found it possible to ferment mashes havingconcentrations up to 9% of molasses with fairly uniform results. Byadding a sufficiently large number of spores, which germinate rapidly toproduce the vegetative cells which are the active agents in thefermentation, the effect of the residual contamination can be largely ifnot entirely overcome, so that this inhibiting influence is eliminatedor becomes insignifi-- cant. The butyl organisms rapidlyuse up thesugar, leaving the acid-forming contamination nothing upon which to act.

It is, of course, essential that all bacteriologic precautions'be takento prevent the entrance of outside contamination at any stage of thisprocess, and for his purpose all tanks, pipe lines, valves, and other itnitrogen to sterilize this together with the I excluding it entirely.

molasses in the 50% solution.

My invention is not limited to tl1e'con-- centration of molasses givenin the example, nor to the proportion of corn gluten given in thisexample, nor necessarily to its use at all, nor do I consider itessential that the seed used" for inoculating the molasses mash shouldbe grown in corn meal or other grain mash, as the results of my workindicate that a molasses mash maybe used in this stage with equally goodresults, Furthermore, I do not wish to be limited to the temperature of63 C. at which to heat the spore seed, since I have successfullyemployed temperatures varying from 57 to 71 C. It will also beunderstood that my invention is not limited to the use of a.pasteurizing temperature onlyat the point of inoculating the finalmash, since this procedure may be used to advantage in preparing thevarious seed stages prior to the final fermentatlon.

The sugar mashes may be made from practically any source of fermentablesugar such as corn sugar, cane sugar, sugar solution obtained byhydrolysis of wood cellulose, molasses, and others, my invention beingparticularly applicable to any fermentable sugar or starch solutioncontaminated by the organism or organisms above described.

The bacillus aceto-butylicum used in the above described process is afaoultative anaerobe, that is, its fermenting action is not materiallyinfluenced by a change in its environment with respect to oxygen.Consequently no effort need be made to insure the presence of air duringthe fermentation. The air is ordinarily excluded to a great extent bythe evolution of gas from the fermenting mass, and of course air ladenitis frequently more/ convenient to avoid contamination'by filtering theair than by Although I'prefer to use for the fermentation ofcarbohydrates pure cultures of bacillus aceto-butylicum, my inventionalso includes the use of cultures obtainable from and in addition to,the bacillus acetobutylicum. a

I. do not limit myself to the fermentation of sugar solutions, such asmolasses, by my new process, as it is equally useful in the fermentationof mashes containing higher polysaccharides, such as starch anddextrine, for example, cereal and potato mashes.

It will be clear from the above description that I use the expressionsterilized mash or sterlized sugar mash to refer to a mash which hasbeen subjected to the ordinary sterilizing treatment, such as heatingwith steam at 20' pounds pressure for one-half hour, and do not mean toimply that the mash so referred to is necessarily entirely free fromcontaminating micro-organisms.- The molasses mash, with which thisprocess is primarily concerned, even after being subjected to" theordinary sterilizing treatment appears to contain, as above indicated, apeculiar micro-organism capable of withstanding for a long time theordinary sterilization temperatures.

I claim 1. The process which comprises adding to a sterilizedfermentable carbohydrate mash a culture of micro-organisms, mainly inthe spore form, which are derivable from ordinary corn meal, aresufficiently heat-resistant to' withstand a temperature of C. for about20' minutes, and are capable of producing butyl alcohol and acetone byfermentation, in the presence of air, of a sterilized corn mealsolution, said addition being made whilesaid mash is at a fermentationtemperature and with said culture at a pasteurization temperatlu-e, andthen allowing fermentation to proceed.

2. The process which comprises adding to a sterlized fermentablecarbohydrate mash a culture of micro-organisms, mainly in the sporeform, which are derivable from ordinary corn meal, are sufficientlyheat-resistant to withstand a temperature of 80 C. for about 20 minutes,and are capable of producing butyl alcohol and acetone by fermentation,in the presence of air, of a sterilized corn meal solution, said.addition be ing made while said mash is at a fermentation tempera ureand immediately after said culture has been heated for a few minutes atapasteurization temperature, and then allowing fermentation to proceed.

3. The process which comprises adding to a sterilized molasses mash aculture of micro-organisms, mainly in thespore form,

protein.

alcohol and acetone by fermentation, in the presence of air, of asterilized corn meal solution, said addition being made while said mashis at a fermentation temperature and with said culture at apasteurization temperature, and then allowing fermentation to" proceed.

4. The process which comprises adding to a sterilized sugar mash aculture of micro-- organisms, mainly in the spore form, which are.derivable from ordinary corn meal, are sufficiently heat-resistant towithstand a temperature of 80 C. for about 20 minutes, and are capableof producing butyl alcohol and acetone by fermentation, in the presenceof air, of a sterilized corn meal solution, said addition being madewhile said mash is at a fermentation temperature and immediately aftersaid culture has beenheated for a few minutes at a temperature of fromabout 57 to 77 C., and then allowing fer-- nientation to proceed.

5. A process as set forth in claim 1 in which the carbohydrate mashcontains a vegetable protein readily assimilated by the micro-organismswhich are to act as the fermenting agents.

(3. A process as set forth in claim 1 in.

which the carbohydrate mash contains corn 7. A process as set forth inclaim 2 in which the carbohydrate mash contains corn protein. i

8. A. process as set forth in claim 3 in which the molasses mashcontains corn protein. 7

S). A process as set forth in claim 4 in which the sugar mash containscorn protein.

10. The process which comprises heating a culture of bacillusaeeto-butylicum existing in the spore form to a pasteurizationtemperature, shortly thereafter, andwhile said culture is still at apasteurization temperature. adding said culture to a fermentablecarbohydrate mash while the latter is at a fermentation temperature,andthen allowing fermentation toproceed to completion.

11. The process which comprises heating a culture of bacillusaceto-butylicum existing in the spore form to .a pasteurizationtemperature, shortly thereafter and while said culture is still at apasteurization temperature, adding said culture to a sterilized molassesmashwhile the latter is at a fermentation temperature, and then allowingfermentation to proceed to completion.

12. The process which comprises heating a culture of bacillusaceto-butylicum, exist ing in the spore form, at a temperature betweenabout 57 and 77 C., adding said culture while at a temperature betweenabout 57 and 77 C. to a sterilized sugar mash maintained at afermentation temperature and containing a vegetable protein readilyassimilated by said bacillus, and then allowing fermentation to procee13. The process which comprises heating -a culture of bacillusaceto-butylicum, existing in the spore form, at a temperature betweenabout 57 and 77 C., adding said culture while at a temperature betweenabout -'57 and 77 C. to a sterilized sugar mash maintained at afermentation temperature and containing corn lowing fermentation toproceed.

14. The process which comprises heating a culture of bacillusaceto-butylicum, existing in the spore form,for about one quarter of'anhour at a temperature of about 63 C., then adding the culture to asterilized molasses mash maintained at a fermentation temperatureandcontaining corn protein, and allowing fermentation to proceed.

15. The process which comprises dividing a sterilized fermentablecarbohydrate mash into two unequal portions, inoculating the smallerportion with a culture of bacillus aceto-butylicum existing in the sporeform, maintaining the inoculated portion for a few minutes at atemperature between.

about 57 and 77 0., and then adding the inoculated portion to the largerportion while the latter is at such a temperature that the mash will beat about a fermentation temperature after the mixing of the two protein,and then al

